Method and device for purifying nucleic acids

ABSTRACT

The invention concerns a method for isolating and purifying nucleic acids from a sample and a device that is suitable therefore.

BACKGROUND OF THE INVENTION

This application claims the benefit of priority under 35 U.S.C. §119 ofEP Application 004006508.8, filed Mar. 18, 2004 and EP Application No.04012677.3 filed May 28, 2004, the contents of which are herebyincorporated by reference.

TECHNICAL FIELD

The invention concerns a method for isolating and purifying nucleicacids from a sample and a suitable device therefore.

BACKGROUND OF INVENTION

The introduction of the polymerase chain reaction (PCR) and subsequentalternative amplification systems for nucleic acids has enabled the useof this genetic material as a specimen for diagnostic tests. As aconsequence new analytical methods are now available especially fordiagnosing hereditary diseases, a predisposition for certain diseasesand infectious diseases which, among others, enable an early diagnosisof the condition.

In order to convert the genetic material into a suitable form forenzymatic amplification, it is necessary to release it from thebiological sample material. In addition the nucleic acid must beprotected from degradation by nucleases from the biological material orthe environment and from degradation by chemical reaction conditions.The most stringent requirements concern the freedom from contaminationof the biological sample and the nucleic acid isolated therefrom. Forthe amplification the nucleic acid should be present in a buffered,aqueous, substantially salt-free solution.

Whereas PCR usually uses very small amounts of analyte (pg-ng range),special problems require the processing of a larger quantity of sample.For example, in order to identify circulating tumour cells at asensitivity of one tumour cell against a background of normal cells, thenucleic acid has to be isolated from 10-20 ml of a blood sample. Afterhomogenizing the sample, an aliquot of the isolated RNA can then beexamined for the expression of a tumour-associated gene.

In addition to the classical methods of nucleic acid isolation by meansof enzymatic, mechanical or chemical lysis of the sample material,subsequent extraction of the proteins and lipids of phenol andphenol/CHCl₃ and precipitation of the nucleic acid from the aqueousphase by ethanol or i-propanol (Sambrook, J., et al., Molecular Cloning,Cold Spring Harbor Laboratory Press, 1989, 2^(nd) edition, 9.16-9.23;Ausubel, F. M., et al., Current Protocols in Molecular Biology, JohnWiley & Sons, 1987, 2.1.1-2.4.5), several commercial kits have beendeveloped especially for PCR sample preparation in recent years whichutilize the property of nucleic acids to bind to glass surfaces underchaotropic salt conditions which has been known since the end of theseventies (Vogelstein, B., et al., Proc. Natl. Acad. Sci. USA 75 (1979)615-619). Other constituents of biological material such as proteins,lipids or salts, are not bound and are therefore separated.Centrifugation vessels with glass fleece inlays or silica gelsuspensions which allow a batch process are known. In addition multipledevices are known in a strip and 96-well microtitre plate format withthe glass fleeces recessed into the base which can be operated with theaid of a vacuum chamber attached underneath as well as bycentrifugation. In these methods the volume of the samples is oftenlimited. Moreover, large amounts of buffer are necessary to effectivelyelute the nucleic acids from the glass fleeces which results in adiluted solution of the isolated molecules and requires additionalpreparation steps for certain applications.

A modified method (Miller et al., Nucl. Acids Res. 16: 1215) uses aconcentrated salt solution to precipitate proteins and otheraccompanying substances after lysis of the sample material. The nucleicacids in the supernatant are then precipitated by ethanol and collectedby centrifugation. After the nucleic acids have been dissolved they canbe used for amplification.

WO 93/11221 discloses a method and a device for isolating and purifyingnucleic acids which uses anion exchangers and mineral carriersubstances. U.S. Pat. No. 5,104,533 discloses a filtration unit withpressure compensation. U.S. Pat. No. 4,270,921 discloses the combinationof a microcolumn and a centrifuge tube. WO 98/32877 discloses a devicefor isolating nucleic acids which is composed of two vessels connectedby a closure element which contains a material for binding nucleicacids. U.S. Pat. No. 4,956,298 discloses a separation or reaction columnconsisting of a centrifuging vessel and a receiving body where thereceiving body contains a column material and the centrifuging vesselcollects the effluent of the receiving body. DE 19512361 discloses amethod for isolating a biological material which uses a compressibleporous matrix to bind the biological material and compresses thematerial in order to elute the material. EP 588564 describes a devicefor affinity separation comprising a capture membrane located in apipette tip. WO 96/41810 discloses the removal of DNA from a cellsuspension with the aid of a hollow membrane filter and an ion exchangestep. The manufacture of a device containing a material for bindingnucleic acids is known from EP 738733. WO 02/053256 discloses a deviceand a method for purification comprising a sample holder with a columninsert portion, whereas a column module is secured in said column insertportion. U.S. Pat. No. 6,177,009 and the German utility models DE 298 03712 U1 and DE 202 18 503 U1 describe a device for treating biomoleculescomprising a separation column which has a separation device and acollecting vessel for the liquid that flows out.

SUMMARY OF THE INVENTION

The object of the present invention was to provide a new device and anew method for purifying or isolating nucleic acids from larger samplevolumes.

The object is achieved according to the invention by a device whoseindividual components are shown in FIG. 1. Furthermore a method isdescribed in which the device according to the invention is used andwhose individual phases are also shown schematically and as an examplein FIG. 1. The method according to the invention in which the deviceaccording to the invention is used ensures a larger yield of nucleicacids.

One subject matter of the present invention is a device for purifying orisolating nucleic acids consisting of a first hollow body 100 with aninlet opening 101 for a sample and an outlet opening 102 and a secondhollow body 200 with an inlet opening 201 and an outlet opening 202,wherein the inlet opening of the second hollow body 201 is functionallyconnected to the outlet opening of the first hollow body 102, the firstand the second hollow body can be detached from one another, a nucleicacid-binding material 203 is located in front of the outlet opening ofthe second hollow body 202, the second hollow body 200 has a smallervolume compared to the first hollow body 100 and the first hollow body100 has a volume of more than 5 ml.

Another aspect of the invention concerns a closable vessel 400 whichcontains a device according to the invention.

The invention also concerns a method for purifying or isolating nucleicacids from a sample comprising the steps:

-   -   a) providing the device according to the invention,    -   b) transferring the sample into the device through the inlet        opening in the first hollow body,    -   c) passage of the sample from the second hollow body through the        nucleic acid-binding material into a vessel during which the        nucleic acids bind to the nucleic acid-binding material,    -   d) optionally washing the nucleic acids bound to the nucleic        acid-binding material,    -   e) detaching the second hollow body from the first hollow body        and transferring the second hollow body into a receiving vessel        900,    -   f) washing the nucleic acids bound to the nucleic acid-binding        material,    -   g) eluting the nucleic acids bound to the nucleic acid-binding        material whereby the nucleic acids are collected in a second        receiving vessel 13 and are thus purified or isolated.

The invention also concerns a kit for purifying or isolating nucleicacids from a sample composed of a device according to the invention or avessel according to the invention and chaotropic reagents for bindingthe nucleic acids to the nucleic acid-binding material.

Another subject matter of the invention is the use of a device accordingto the invention or of a vessel according to the invention to purify orisolate nucleic acids from a sample.

A nucleic acid-binding material is understood as a material to whichnucleic acids bind non-covalently under certain conditions whereas othersubstances in a sample do not bind under these conditions. This nucleicacid binding is reversible and hence the nucleic acids can besubsequently eluted again from the material by changing the conditions.

A matrix is understood within the scope of this invention as a materialin which particles or fibres of the nucleic acid-binding material areembedded. This matrix material is permeable to liquids so that thesample can pass through the matrix, the nucleic acids can make contactwith the nucleic acid-binding material and other components of thesample can leave the matrix. Solid materials having a small diameter arereferred to as particles by a person skilled in the art. These particlespreferably have an essentially spherical surface. Lamellar andfilamentary particles made of nucleic acid-binding material are referredto as fibres.

Within the scope of this invention a hollow body is a hollow structurewith an inlet opening through which a sample can enter the hollow bodyand an outlet opening through which the sample can again leave thehollow body. In contrast a vessel is a hollow structure with only oneinlet opening through which a sample can enter the vessel. Hence it canbe used to collect a sample.

Within the scope of this invention a functional connection of hollowbodies is understood to mean that the two hollow bodies are connected insuch a manner that it is possible to carry out the method according tothe present invention. For this purpose it should be possible to detachthe connection as required, it should be impermeable to liquids and, forcertain applications, it should also prevent air exchange with theenvironment. Furthermore it should ensure a passage of the sample fromthe first hollow body into the second hollow body without loss.

Chaotropic reagents are understood as substances which change thesecondary, tertiary and/or quarternary structure of proteins or nucleicacids but do not affect at least their primary structure. Examples areguanidinium thiocyanate, guanidinium hydrochloride, NaI, KI, sodiumthiocyanate or combinations of these substances. Within the scope ofthis invention chaotropic reagents are understood as all chemicalsubstances which disturb the ordered structure of liquid water and thuscause DNA or RNA to bind from this aqueous solution to a glass surface.Other substances such as NaCl, KCl or CaCl2 may be present in thesolution in order to modify the ionic strength. The property of DNA andRNA to bind under chaotropic conditions to glass surfaces can beutilized to isolate them from a solution containing other biologicalmaterials since the binding to the glass surface is reversible. If, forexample, the concentration of the chaotropic reagents is reduced or thechaotropic reagents are entirely removed, the DNA or RNA can be elutedagain.

DESCRIPTION OF THE FIGURES

FIG. 1: Schematic representation of the method according to theinvention shown as an example using the device according to theinvention.

FIG. 2: Alternative embodiment of the device according to the inventionin which means are provided that enable a pressure difference to begenerated and thus ensure passage of the sample through the nucleicacid-binding material.

DETAILED DESCRIPTION OF THE INVENTION

One subject matter of the present invention is a device for purifying orisolating nucleic acids consisting of a first hollow body 100 with aninlet opening 101 for a sample and an outlet opening 102 and a secondhollow body 200 with an inlet opening 201 and an outlet opening 202,wherein the inlet opening of the second hollow body 201 is functionallyconnected to the outlet opening of the first hollow body 102, the firstand the second hollow body can be detached from one another, a nucleicacid-binding material 203 is located in front of the outlet opening ofthe second hollow body 202, the second hollow body 200 has a smallervolume compared to the first hollow body 100 and the first hollow body100 has a volume of more than 5 ml.

Materials that bind nucleic acids are known to a person skilled in theart. The material can be particulate as well as fibrous. If the materialis composed of particles it has proven to be advantageous to immobilizethese particles for example by incorporating them betweenliquid-permeable lamellae e.g. fabrics or fleeces made of a fibrousmaterial such as cellulose or plastics which have such narrow pores thatthe particles are held between the lamellae. The nucleic acid-bindingmaterial is preferably mainly composed of silicon dioxide or containssilicon dioxide in the form of fibres or particles. The nucleicacid-binding material is particularly preferably a glass fleece or asilica gel or it is composed of zeolite. The nucleic acid-bindingmaterial is also preferably composed of metal oxides or mixed metaloxides in the form of fibres or particles. The nucleic acid-bindingmaterial is particularly preferably composed of aluminium oxide, hafniumoxide or zirconium oxide or contains aluminium oxide, hafnium oxide orzirconium oxide in the form of fibres or particles.

The nucleic acid-binding material is preferably a fibrous material e.g.in the form of fabrics or fleeces. Suitable materials are for exampleknown from methods for isolating nucleic acids with the aid ofcentrifugation tubes (EP 738733) or multiple devices in a strip format(EP 616638). The nucleic acid-binding material must have the propertythat the sample liquid can pass through the material without anyadditional action of force or by applying force e.g. by applyingpressure or underpressure. However, since the nucleic acids are notbound in the present method by filtration of the nucleic acids from thesample but by a method which utilizes the affinity of nucleic acids tosurfaces, it is possible to use a relatively coarsely porous material.This facilitates the flow even of relatively viscous sample liquids.

The liquid-permeable, nucleic acid-binding material is able to bindnucleic acids but allows passage of the surrounding liquid and othercomponents dissolved therein such as proteins etc. In a first variantthe nucleic acids can be bound sequence-specifically by capture probesattached to the surface of the material. The capture probes have a basesequence which can bind to a complementary base sequence in the nucleicacids to be isolated under hybridizing conditions. The use ofsequence-specific materials allows the selective isolation of nucleicacids of a particular sequence. A method for binding nucleic acids topeptidic nucleic acids on the surface of solids is described for examplein WO 95/14708.

Zirconium oxide, hafnium oxide or aluminium oxide (EP 897978) and alsotitanium oxide are for example suitable as the nucleic acid-bindingmaterial. The hydrated surfaces of these materials have sufficientpositive charges to bind negatively charged nucleic acids. The surfaceof the oxides can be hydrated in basic solutions. The nucleic acids canthen be subsequently eluted by washing with low-molecular alcohols orother wash solutions having a low pH.

In a preferred embodiment the liquid-permeable, nucleic acid-bindingmaterial has a surface containing glass. The property of glass to bindnucleic acids in a particulate and fibrous form has been known for along time. Chaotropic reagents such as guanidinium thiocyanate,guanidinium hydrochloride, NaI, KI, sodium thiocyanate or combinationsof these substances are necessary for the reversible binding to glasssurfaces (U.S. Pat. No. 5,234,809). DE-A-19512369 describes the use ofglass fleeces to isolate nucleic acids. A method is proposed inEP-B-0389 063 in which the sample is mixed with a mixture of achaotropic guanidinium salt and silica particles. Under these conditionsnucleic acids bind sequence-independently to the silica surface. Theother sample components can be washed away and the nucleic acids can besubsequently eluted in an aqueous buffer.

Nucleic acids within the sense of the invention are understood asnucleic acids of any origin e.g. nucleic acids of viroid, viral,bacterial or cellular origin. If the nucleic acids are not readilyaccessible in the sample, they are preferably made accessible withappropriate reagents. This includes changing the pH (alkaline), heat,repeated extreme changes in temperature (freezing/thawing), changing thephysiological growth conditions (osmotic pressure), action ofdetergents, chaotropic salts or enzymes (e.g. proteases and lipases).Sample materials from which nucleic acids can be released in this mannerare in particular cell-containing media, cell smears and tissuesections. The nucleic acids can be RNA as well as DNA.

The device according to the invention consists of two hollow bodies100/200 which are connected by a functional connection 3 and each ofwhich has an inlet opening 101/201 and an outlet opening 102/202 for asample. This device according to the invention is preferably shaped suchthat it at least partially fits into a vessel 400. In this contextfunctionally connected means that the two hollow bodies are connected insuch a manner that it is possible to carry out the method according tothe present invention. For this purpose it should be possible to detachthe connection as required, it should be impermeable to liquids and, forcertain applications, it should also prevent air exchange with theenvironment. Furthermore it should ensure a passage of the sample fromthe first hollow body into the second hollow body without loss. Thesehollow bodies are preferably essentially cylinders. The openings arepreferably essentially round. In addition to a cylindrical portion atthe inlet opening, the first hollow body particularly preferably alsohas a section that narrows conically towards its outlet opening. Thefirst hollow body preferably has a volume of more than 5 ml. Once thesample 5 has passed through the outlet opening 102 of the hollow body100, it enters the second hollow body 200 without loss through thepreferably round inlet opening 201 by means of the functional connection3.

The liquid permeable, nucleic acid-binding material 203 is located inthe second hollow body. The nucleic acid-binding material is disposed atthe preferably round outlet opening 202 of the hollow body in such amanner that the emerging sample liquid 5 has to pass through the nucleicacid-binding material. In this process the nucleic acids are bound tothe nucleic acid-binding material.

The second hollow body can be similar to the first hollow body withregard to its design as well as material. In particular the hollowbodies should be able to receive at least the volume of the sampleliquid 5. The second hollow body preferably has a smaller volumecompared to the first hollow body. This hollow body is preferably shapedsuch that it at least partially fits into a receiving vessel 900. Inaddition the second hollow body consists of a material and has a size sothat it is suitable for centrifugation at high speeds of up to 14,000revolutions per minute.

Both hollow bodies have means that enable them to be functionally andreversibly connected together. The functional connection 3 of the firstand second hollow body is preferably a screw connection i.e. for examplean outer or inner screw thread. If the first hollow body for example hasa thread, the means of the second hollow body is a matching counterthread. In another preferred embodiment of the present invention thefirst and the second hollow body are connected together by pressingforces e.g. by means of a plug connection.

The two hollow bodies preferably together have a volume which allowsthem to receive the entire sample and other reagents e.g. to facilitatethe binding of nucleic acids to the nucleic acid-binding material. Thevolume is more than 500 microlitres, preferably between 1 and 1000 ml,particularly preferably between 1 and 100 ml and most preferably between5 and 50 ml or 10 and 30 ml. A volume of 10, 20 or 30 ml is particularlypreferred. The first hollow body has a preferred volume between 1 and100 ml, particularly preferred between 5 and 50 ml, most preferredbetween 10 and 30 ml. The second hollow body has preferably a volume ofbelow 2 ml, particularly preferred between 1 and 2 ml.

A preferred variant of the device according to the present invention isa device characterized in that the two hollow bodies together have avolume of between 5 and 50 ml.

Furthermore the first hollow body preferably has means near to its inletopening 101 for holding 103 the hollow body in a vessel 400 such thatits position is fixed. This vessel has an inlet opening 401 which ispreferably round. The vessel preferably has a cylindrical portion at theinlet opening 401 and a conical region at its end. The vessel is used toreceive the liquid 8 which emerges from the second hollow body 200 afterit has passed through the nucleic acid-binding material 203. The vesselcan preferably be closed.

The vessel 400 is particularly preferably shaped and provided withclosure means 6 in such a manner that it can completely receive bothfunctionally connected hollow bodies 100/200 and the vessel 400 can beclosed with a closure element 7. The closure element is preferably ascrew cap made of plastic. A circular flange is preferred as the meansfor holding 103 the first hollow body in the vessel 400 which protrudesbeyond the edge of the vessel such that the hollow body rests on it andcannot penetrate further into the vessel. This circular flange ispreferably shaped such that it is clamped with the vessel by the closureelement 7 and thus acts as a seal. In this case the closure element ispreferably a screw cap that is placed from above and the vessel then hasthe corresponding counter thread.

Alternatively the vessel can have a section that narrows conically atits inlet opening 401 such that the first hollow body cannot penetratefurther into the vessel at a defined position even without additionalmeans for holding it. In this case the vessel can also be closed with aclosure element. It is also conceivable that the two functionallyconnected hollow bodies only partially fit into the vessel in which caseat least the outlet opening of the second hollow body must completelyextend into the vessel.

In a further preferred variant of the device according to the inventionwhich is shown in FIG. 2, the vessel 400 is provided with means forapplying a pressure difference. This pressure difference can execute orsupport the passage of the sample through the nucleic acid-bindingmaterial. For this purpose the vessel is provided with a connectingpiece 16 for applying overpressure or underpressure and the closureelement 7 is provided with a pressure-equalizing connection 17. In thisvariant of the device it is necessary that the site connecting the twohollow bodies is also impermeable to gases since otherwise a pressuredifference cannot build up across the nucleic acid-binding material.

The second hollow body 200 is preferably shaped such that it at leastpartially fits into a receiving vessel 900. This receiving vesselpreferably has an essentially round inlet opening 901. The receivingvessel 900 is preferably closable and consists of a cylindrical portionat the inlet opening and a conical portion at its end. The receivingvessel is particularly preferably designed such that the second hollowbody 200 provided with holding means 204 fits into it and that thevessel can be closed with a closure element 10. Like the first hollowbody 100, the second hollow body also preferably has a circular flangeas the holding means 204 which extends over the edge of the receivingvessel. When using a screw cap 10 with a thread 11 the circular flangeis clamped with the receiving vessel and acts as a seal. Also in thiscase the receiving vessel may also have a conical shape in order to holdthe second hollow body at a defined position in which the hollow bodycompletely or partially penetrates into the receiving vessel.

In a further variant of the present invention the receiving vessel ispreferably provided with means for applying a pressure difference. Thispressure difference can execute or support the removal of residualliquid from the fleece and the elution of the nucleic acids from thematerial. For this purpose the receiving vessel is provided with aconnecting piece for applying an overpressure or underpressure and theclosure element is provided with a pressure-equalizing connection.

The first or the second hollow body, the vessel or the receiving vesselof the device according to the invention are made of materials that donot bind nucleic acids. The first or the second hollow body, the vesselor the receiving vessel of the device according to the invention arepreferably made of plastic, metal or composite material. Plastics suchas polypropylene, polystyrene, polyethylene or Luran® are particularlypreferred. These have the advantage that they can be easily manufacturedin a multiple injection process and having a high mechanical stabilityunder the conditions of the isolation method according to the invention.

Plastics that can be injection moulded are particularly preferred asmaterials for the hollow body because they already allow theintroduction of the nucleic acid-binding, liquid-permeable materialduring the manufacture of the second hollow body. Especially in the caseof glass fibre fleeces the material can already be permanently pouredinto the closure element already during the injection moulding process.However, it is also possible to only attach the material after themanufacture of the second hollow body in the injection moulding processe.g. by gluing, welding or by fixation with a clamp ring. Processes formanufacturing a hollow body containing nucleic acid-binding material aredescribed in the patent document EP 0 738 733. Such separation columnscontaining nucleic acid-binding material are also commercially available(High Pure™ column from Roche Diagnostics GmbH, Mannheim).

The invention also concerns a closable vessel which contains a deviceaccording to the invention. The closable vessel is preferable shapedsuch that it is able to receive the entire inventive connection of thetwo hollow bodies and can be closed by means of a closure element. Theclosable vessel is particularly preferably a commercial centrifugationvessel such as a plastic tube (Falcon tube) which has a filling volumeof 50 ml and can be closed by means of a screw cap.

Another aspect of the present invention is a method for purifying orisolating nucleic acids from a sample comprising the steps:

-   -   a) providing the device according to the invention,    -   b) transferring the sample into the device through the inlet        opening in the first hollow body,    -   c) passage of the sample from the second hollow body through the        nucleic acid-binding material into a vessel during which the        nucleic acids bind to the nucleic acid-binding material,    -   d) optionally washing the nucleic acids bound to the nucleic        acid-binding material,    -   e) detaching the second hollow body from the first hollow body        and transferring the second hollow body into a receiving vessel        900,    -   f) washing the nucleic acids bound to the nucleic acid-binding        material,    -   g) eluting the nucleic acids bound to the nucleic acid-binding        material whereby the nucleic acids are collected in a second        receiving vessel 13 and are thus purified or isolated.

A preferred variant of the method of the present invention uses a deviceaccording to the invention with a nucleic acid-binding material which ismainly composed of silicon dioxide or contains silicon dioxide in theform of particles or fibres or is particularly preferably a glass fleeceor silica gel or consists of zeolite and a sample to which chaotropicreagents are already added through the inlet opening of the first hollowbody before transfer into the device such that the concentration of thechaotropic reagents is between 1 M and 8 M.

A preferred embodiment of the method according to the invention that isbased on the device according to the invention is described in thefollowing. Firstly the cells of 5 to 30 ml whole blood, serum, plasma orother body fluids are lysed, disrupted and the reagents that areadditionally required e.g. a chaotropic salt or/and protease are addedto the sample liquid. In addition the device according to the inventionis placed in a vessel for collecting the nucleic acid-free liquid (seestep I and II in FIG. 1). After the sample 5 has been transferred intothe device through the inlet opening of the first hollow body, thevessel is preferably tightly closed with a cap (step III in FIG. 1.

In the next step the sample liquid is passed through the nucleicacid-binding material (step IV in FIG. 1). The sample can be passedthrough either already by means of gravitational force or alsopreferably by centrifuging the device. The sample can also be preferablypassed through the nucleic acid-binding material by applying a pressuredifference. In this embodiment of the invention (see FIG. 2) the deviceis additionally provided with means e.g. two connecting pieces 16/17 forapplying the pressure. One connecting piece for applying theoverpressure or underpressure is preferably located on the vessel itselfand the second connecting piece is located on the closure element of thevessel to serve as a pressure-equalizing connection.

During passage of the sample liquid through the nucleic acid-bindingmaterial, the nucleic acids present in the sample are bound to thenucleic acid-binding material while other sample components togetherwith the liquid 8 pass into the vessel. The isolated nucleic acids arenow in the nucleic acid-binding material of the second hollow bodywhich, if required, can be separated from the first hollow body andprocessed further in any desired manner.

Since certain quantities of liquids containing contaminants stillusually adhere to the liquid-permeable material even aftercentrifugation, it is possible to remove substances that are stilladhering by an optional washing step in order to isolate particularlypure nucleic acids before the device is removed from the vessel and thefunctional connection of the hollow bodies is separated. For thispurpose the washing fluid can for example be added through the inletopening of the first hollow body so that it rinses the material to whichthe nucleic acids are bound while it passes through and is thencollected in the vessel. The washing step can preferably be carried outapplying a pressure difference or by centrifuging the device.

In order to detach the nucleic acids again from the liquid-permeable,nucleic acid-binding material, the second hollow body can be removedfrom the device (step V in FIG. 1). For this purpose the second hollowbody is preferably connected to a receiving vessel 900 after it has beenseparated from the first hollow body in order to firstly remove residualliquid 12 present in the fleece (step VI in FIG. 1). This washing ispreferably carried out by centrifuging the device since the secondhollow body connected to the receiving vessel can be subjected to veryhigh centrifugal forces due to its small dimensions. Subsequently thesecond hollow body is connected to a second receiving vessel 13 whichshould collect the eluate (step VII in FIG. 1). The elution liquid 14 iscomposed such that it abolishes the binding of the nucleic acids to thenucleic acid-binding material. The conditions under which the nucleicacids can be detached again depend on the material that is used and theprocess can again be supported by applying a pressure difference or bycentrifugation (step VIII in FIG. 1).

The elution is preferably carried out by centrifuging since this enablesthe nucleic acids to be dissolved in very small volumes of elutionliquid and also allows a reduction in the amount of nucleic acidsremaining in the nucleic acid-binding material. The centrifugation forthe elution and washing is preferably carried out using largercentrifugal forces than the centrifugation for the passage of the sampleand the optional washing. The centrifugation for elution and washing ispreferably carried out at more than 5000 g and the centrifugation forsample passage and optionally washing is preferably carried out at lessthan 5000 g. The second hollow body and the receiving vessel areparticularly preferably shaped such that they can be centrifugedtogether e.g. in an Eppendorf centrifuge (Eppendorf, Hamburg, Germany)at more than 10,000 g. This is possible since the receiving vessel onlyhas to have a considerably smaller volume than the first vessel whichcan only be subjected to a considerably lower centrifugal force (e.g. ina Beckman bench centrifuge (Beckman Coulter, Inc., USA) at about 3000 g)and would thus require more elution liquid. Hence the device accordingto the invention is not only suitable for isolating nucleic acids butalso for transferring nucleic acids from a larger 5 into a smallervolume 15.

The volume of the device for purifying or isolating nucleic acids is animportant feature for the present invention. On the one hand, the volumeof the device must be large in order to apply diluted, voluminoussamples and on the other hand, the size of the device must be small inorder to realize a lysis procedure with small amounts of lysis buffer.The necessary amount of lysis buffer for the efficient elution of thenucleic acid is, among others, depending on the size of the nucleicacid-binding material and on the supporting force of the lysisprocedure. The reversible coupling of a large first hollow body with asmall second hollow body comprising the nucleic acid-binding materialsolves both said requirements. First, the overall volume of both hollowbodies provides the applicability of diluted, voluminous samples duringthe binding process. Second, after the binding process the small secondhollow body can be removed from the device and the nucleic acid may beeluted with a much higher centrifugation force as possible with a stateof the art device applicable for the same initial sample volume.

Another aspect of the invention is a kit for purifying or isolatingnucleic acids from a sample which is composed of a device according tothe invention or a vessel according to the invention and chaotropicreagents for binding the nucleic acids to the nucleic acid-bindingmaterial. The kit can additionally contain other plastic parts that arerequired to carry out the method according to the invention such asmicrotitre plates or simple reaction vessels such as Eppendorf reactionvessels (Eppendorf, Hamburg, Germany). In addition the kit can containfurther reagents that are necessary for the method according to theinvention e.g. lysis buffer containing chaotropic reagents, detergent,alcohol or mixtures of these substances that lyse cells, washing buffercontaining chaotropic reagents and/or alcohol or buffer with acidic pHto wash the nucleic acid-binding material to which the nucleic acids arebound or elution buffer which enables the nucleic acids to be detachedfrom the nucleic acid-binding material. According to the invention thesecomponents of the kit can be provided individually or in storagecontainers. The reagents are usually offered ready-to-use but can alsobe sold in the form of stock solutions that have to be diluted beforeuse.

The invention also concerns the use of a device according to theinvention or a vessel according to the invention to purify or isolatenucleic acids from a sample.

Specifically the invention encompasses the following aspects:

-   1. Device for purifying or isolating nucleic acids consisting of a    first hollow body 100 with an inlet opening 101 for a sample and an    outlet opening 102 and a second hollow body 200 with an inlet    opening 201 and an outlet opening 202 wherein the inlet opening of    the second hollow body 201 is functionally connected to the outlet    opening of the first hollow body 102, the first and the second    hollow body can be detached from one another and a nucleic    acid-binding material 203 is located in front of the outlet opening    of the second hollow body 202.-   2. Device according to item 1, wherein the second hollow body has a    smaller volume compared to the first hollow body.-   3. Device according to one of the items 1 or 2, wherein the device    is shaped such that it at least partially fits into a vessel 400.-   4. Device according to one of the items 1 to 3, wherein the second    hollow body is shaped such that it at least partially fits into a    receiving vessel 900.-   5. Device according to one of the items 1 to 4, wherein the first    and the second hollow body are connected together by a screwed    joint.-   6. Device according to one of the items 1 to 4, wherein the first    and the second hollow body are connected together by pressing    forces.-   7. Device according to one of the items 1 to 6, wherein the first    and/or the second hollow body are essentially cylinders.-   8. Device according to item 7, wherein the first hollow body in    addition to its cylindrical section at the inlet opening, has a    section that becomes conically narrower towards its outlet opening.-   9. Device according to one of the items 1 to 8, wherein the openings    of the first and/or of the second hollow body are essentially round.-   10. Device according to one of the items 1 to 9, characterized in    that the first hollow body has a volume of more than 5 ml.-   11. Device according to one of the items 3 to 10, wherein the vessel    is closable.-   12. Device according to one of the items 3 to 11, wherein the vessel    consists of a cylindrical section at the inlet opening and a conical    section at its end.-   13. Device according to one of the items 3 to 12, wherein the vessel    has means for applying a pressure difference.-   14. Device according to one of the items 4 to 13, wherein the    receiving vessel is closable.-   15. Device according to one of the items 4 to 14, wherein the    receiving vessel is closable and consists of a cylindrical section    at the inlet opening 401 and a conical section at its end.-   16. Device according to one of the items 4 to 15, wherein the    receiving vessel has means for applying a pressure difference.-   17. Device according to one of the items 4 to 16, wherein the inlet    openings of the vessel 401 and/or of the receiving vessel 901 are    essentially round.-   18. Device according to one of the items 1 to 17, characterized in    that the nucleic acid-binding material is mainly composed of silicon    dioxide or contains silicon dioxide in the form of fibres or    particles.-   19. Device according to one of the items 1 to 18, characterized in    that the nucleic acid-binding material is a glass fleece or a silica    gel or is composed of zeolite.-   20. Device according to one of the items 1 to 19, characterized in    that the nucleic acid-binding material is composed of metal oxides    or mixed metal oxides or contains metal oxides or mixed metal oxides    in the form of fibres or particles.-   21. Device according to one of the items 1 to 20, characterized in    that the nucleic acid-binding material is composed of aluminium    oxide, hafnium oxide or zirconium oxide or aluminium oxide, hafnium    oxide or zirconium oxide in the form of fibres or particles.-   22. Device according to one of the items 1 to 21, characterized in    that the first or the second hollow body, the vessel or the    receiving vessel are made of a material that does not bind nucleic    acids.-   23. Device according to one of the items 1 to 22, characterized in    that the first or the second hollow body, the vessel or the    receiving vessel is composed of plastic, metal or a composite    material.-   24. Device according to item 23, characterized in that the second    hollow body is made of polypropylene.-   25. Closable vessel 400 which contains a device according to one of    the items 1 to 24.-   26. Method for purifying or isolating nucleic acids from a sample by    -   a) providing a device according to one of the items 1 to 25,    -   b) transferring the sample into the device through the inlet        opening in the first hollow body,    -   c) passage of the sample from the second hollow body through the        nucleic acid-binding material into a vessel during which the        nucleic acids bind to the nucleic acid-binding material,    -   d) optionally washing the nucleic acids bound to the nucleic        acid-binding material,    -   e) detaching the second hollow body from the first hollow body        and transferring the second hollow body into a receiving vessel        900,    -   f) washing the nucleic acids bound to the nucleic acid-binding        material    -   g) eluting the nucleic acids bound to the nucleic acid-binding        material whereby the nucleic acids are collected in a second        receiving vessel 13 and are thus purified or isolated.-   27. Method according to item 26, characterized in that a device as    claimed in one of the items 18 or 19 is used and that chaotropic    reagents are additionally added to the sample through the inlet    opening in the first hollow body before transfer into the device    such that the concentration of the chaotropic reagents is between 1    M and 8 M.-   28. Method according to item 26, characterized in that the passage    of the sample in step c) , the optional washing in step d), the    washing in step f) or the elution in step g) is carried out by    applying a pressure difference.-   29. Method according to item 26, characterized in that the passage    of the sample in step c) ,the optional washing in step d), the    washing in step f) or the elution in step g) is carried out by    centrifugation.-   30. Method according to item 29, characterized in that the    centrifugation in steps f) and g) is carried out at a larger    centrifugal force compared to the centrifugation in steps c) and d).-   31. Method according to item 29, characterized in that the    centrifugation in steps c) and d) is carried out with a centrifugal    force of less than 5000 g.

32. Method according to item 29, characterized in that thecentrifugation in steps f) and g) is carried out with a centrifugalforce of more than 5000 g.

-   33. Kit for purifying or isolating nucleic acids from a sample    composed of    -   a) a device according to one of the items 1 to 24 or a vessel        according to item 25,    -   b) chaotropic reagents for binding the nucleic acids to the        nucleic acid-binding material.-   34. Use of a device according to one of the items 1 to 24 or a    vessel according to item 25 to purify or isolate nucleic acids from    a sample.

The invention is further elucidated by the following examples,publications and figures the protective scope of which is derived fromthe patent claims. The described methods are to be understood asexamples which still describe the invention even after modifications.

EXAMPLES Example 1

The following example of a DNA isolation from 5 ml serum is intended tofurther elucidate the present invention. The so-called “UpScaleHighPure” method utilizes the so-called “UpScale HighPure” kit which iscomposed of a commercially available “HighPure™ Column” (RocheDiagnostics GmbH, Mannheim, Germany), a pluggable volume attachment andreagents that are identical to the reagents of the “MagNA Pure® LC TotalNucleic Acid Isolation Kit—Large Volume” (Roche Diagnostics GmbH,Mannheim, Germany). The volume attachment (made of polypropylene) has acylindrical shape in this example (inner diameter 2.5 cm, length 5 cm)with a filling volume of about 25 ml, a round inlet opening and anoutlet opening in which a HighPure column (volume about 1.5 ml) can beattached. An Eppendorf centrifuge (Eppendorf, Hamburg, Germany), aBeckman centrifuge (Beckman Coulter, Inc., USA) with 50 ml Falcon tubes,a commercial vortexer and Eppendorf reaction vessels (Eppendorf,Hamburg, Germany) are also used for the DNA isolation by means of the“UpScale HighPure” method.

Procedure for the DNA Isolation

250 μl of a 40 mg/ml proteinase K solution are placed in a 50 ml Falcontube. 5 ml sample (serum or plasma) is added, vortexed and thenincubated for 10 min at room temperature. In the next step, 6.25 mllysis/binding buffer are added, vortexed and the solution is incubatedfor 10 min at 65° C. This is followed by a centrifugation at 1900 g(removal of foam). In the next step 3.125 ml isopropanol are added,mixed and centrifuged for 1 min at 1900 g, then the mixture is allowedto stand for 10 min at room temperature. In the next step the mixture isapplied in one portion (ca. 15 ml) to the volume attachment of thedevice, the remaining residual liquid is also applied to the column bypipetting. It is firstly centrifuged for 2 min at 1900 g (includingacceleration), subsequently for 1 min at 3300 g. In the next step theeluate is discarded.

This is followed by various washing steps (the used washing buffers arecommercially available “MagNA Pure® LC Total Nucleic Acid IsolationKit—Large Volume”, cat. No. 3264793, Roche Diagnostics GmbH, Mannheim,Germany): Firstly the device is placed in a new 50 ml Falcon tube, 2 mlwashing buffer 1 is added and centrifuged for 2 min at 3300 g.Afterwards 2 ml washing buffer 2 is added and centrifuged for 2 min at3300 g. In the next washing step 2 ml washing buffer 3 is added andcentrifuged for 2 min at 3300 g (the 3 washing steps can be carried outwithout changing the Falcon tube). The volume attachment is now removedfrom the High Pure column and the High Pure column is placed in anEppendorf cup, closed with a cap and centrifuged in an Eppendorfcentrifuge for 1 min at 20,000 g. This removes residual liquid from thefleece.

The next step comprises eluting the DNA. 50-100 μl elution buffer isapplied to the fleece and the High Pure column is closed with a cap.Afterwards it is incubated for 3 min at room temperature andsubsequently centrifuged for 1 min at 20,000 g in the Eppendorfcentrifuge. The Eppendorf reaction vessel contains the eluate and theHighPure™ column can be discarded.

Example 2

The following example shows a comparison of a DNA isolation according tothe inventive method and an isolation according to the state of the art.A serum sample is prepared analogously to example 1 and evenly dividedbetween 2 HighPure™ columns with a volume attachment. The experimentalprocedure for both columns is identical up to the step of removingresidual liquid. In one of the columns the volume attachment isseparated from the HighPure column in the following, the residual liquidin the fleece is removed by centrifugation with the aid of an Eppendorfcentrifuge (about 15 μl) and the bound nucleic acid is subsequentlyeluted in 50 μl with the aid of the Eppendorf centrifuge.

In contrast in the second column the connection between the HighPurecolumn and the volume attachment is retained, the entire device isinserted in a Falcon tube and the elution is carried out with 50 μlsolution in a Beckman centrifuge (2 min 3300 g), without previouslyremoving the residual liquid from the fleece.

Results show that on average the DNA yield was about 30% lower over 10experiments without separating the device and without the Eppendorfcentrifuge.

Example 3

The following example shows an other comparison of a DNA isolationaccording to the inventive method and an isolation according to thestate of the art. A serum sample is prepared analogously to example 1and evenly divided between a HighPure™ column with volume attachment anda “NucleoSpin™ Funnel” column from Macherey & Nagel, Düren, Germany(Cat.-No. 740959). The experimental procedure for both columns isidentical up to the step of removing residual liquid. In case of theHighPure™ column the volume attachment is separated from the HighPurecolumn in the following, the residual liquid in the fleece is removed bycentrifugation with the aid of an Eppendorf centrifuge (about 15 μl) andthe bound nucleic acid is subsequently eluted in 60 μl with the aid ofthe Eppendorf centrifuge.

In contrast the column of Macherey & Nagel is inserted in a Falcon tubeand the elution is carried out with 60 μl solution in a Beckmancentrifuge (2 min 3300 g), without previously removing the residualliquid from the fleece.

Results show that on average the DNA yield using a HighPure™ column withvolume attachment was about a factor of 3 to 4 better over 5 differentserum probes (3 determinations per probe) than in case of using thecolumn of Macherey & Nagel.

Example 4

The 5 serum probes used in example 3 were also used to compare theperformance of the inventive device with the performance of twocommercial precipitation kits for DNA isolation or purification, the“RTP® DNA/RNA Virus Supersense” kit of Invitek GmbH, Berlin, Germany(Cat-No. 10404002, Lot OC030036) and the “QIAmp® UltraSens™” kit fromQiagen GmbH, Hilden, Germany (Cat-No. 53704, Lot 11862713). Both kitswere used according to the protocols of the manufactures (Invitek: Vers.TH11/03.2 March 2003; Qiagen: January 2003), whereas in case of theQiagen-kit the protocol was adapted from 1 ml to 5 ml volume.

In comparison with the HighPure® column adapted for large volumes, theQiagen-kit isolated an average amount of DNA that was reduced by afactor of around 9, whereas the Invitek-kit isolated an average amountof DNA that was reduced even by a factor of around 80.

LIST OF REFERENCES

-   Ausubel, F. M., et al., Current Protocols in Molecular Biology, John    Wiley & Sons, 1987, 2.1.1-2.4.5-   DE 19512361-   DE-A-19512369-   DE 202 18 503 U1-   DE 298 03 712 U1-   EP 588564-   EP 616638-   EP 738733-   EP 897978-   EP-B-389063-   Miller, S. A., et al., Nucleic Acids Res. 16 (1988) 1215-   Sambrook, J., et al., Molecular Cloning, Cold Spring Harbor    Larboratory Press, 1989, 2^(nd) edition, 9.16-9.23-   U.S. Pat. No. 4,270,921-   U.S. Pat. No. 4,956,298-   U.S. Pat. No. 5,104,533-   U.S. Pat. No. 5,234,809-   U.S. Pat. No. 6,177,009-   Vogelstein, B., et al., Proc. Natl. Acad. Sci. USA 76 (1979) 615-619-   WO 93/11221-   WO 95/14708-   WO 96/41810-   WO 98/32877-   WO 02/053256

1. A device for purifying or isolating nucleic acids comprising: a firsthollow body with an inlet opening for receiving a sample and an outletopening, a second hollow body with an inlet opening and an outletopening, wherein the inlet opening of the second hollow body isfunctionally connected to the outlet opening of the first hollow body,the first hollow body and the second hollow body can be detached fromone another, a nucleic acid-binding material is located inside thesecond hollow body and above the outlet opening of the second hollowbody, the second hollow body has a smaller volume compared to the firsthollow body and the first hollow body has a volume more than 5 ml, avessel for receiving the first hollow body and the second hollow body,wherein the vessel comprises a cylindrical section at its inlet openingand a conical section at its end, and a receiving vessel with a sizesufficient to enclose the second hollow body, wherein the receivingvessel comprises means for applying a pressure to the device.
 2. Thedevice as claimed in claim 1, wherein the device at least partially fitsinto a vessel.
 3. The device as claimed in claim 1, wherein the firsthollow body and the second hollow body are connected together bypressing forces.
 4. The device as claimed in claim 1, wherein thenucleic acid-binding material comprises silicon dioxide.
 5. The deviceas claimed in claim 4, wherein the silicon dioxide is in the form offibres or particles.
 6. The device as claimed in claim 1, wherein thefirst and second hollow body together have a volume between 5 and 50 ml.7. The device as claimed in claim 1, wherein the first hollow body andthe second hollow body are connected together by a screwed joint.
 8. Thedevice as claimed in claim 1, wherein the first hollow body and/or thesecond hollow body are/is cylinders or cylinder.
 9. The device asclaimed in claim 8, wherein the first hollow body comprises a sectionthat becomes conically narrower towards its outlet opening.
 10. Thedevice as claimed in claim 1, wherein the openings/opening of the firsthollow body and/or the second hollow body are/is round.
 11. The deviceas claimed in claim 1, wherein the vessel for receiving the first hollowbody and the second hollow body is closable.
 12. The device as claimedin claim 11 claim 1, wherein the receiving vessel is closable.
 13. Thedevice as claimed in claim 1, wherein the receiving vessel comprises acylindrical section at its inlet opening and a conical section at itsend.
 14. The device as claimed in claim 1, wherein the inletopenings/opening of the vessel for receiving the first hollow body andthe second hollow body and/or the receiving vessel are/is round.
 15. Thedevice as claimed in claim 1, wherein the nucleic acid-binding materialis selected from the group consisting of glass fleece, silica gel andzeolite.
 16. The device as claimed in claim 1, wherein the first hollowbody or the second hollow body or the vessel for receiving the firsthollow body and the second hollow body or the receiving vessel comprisesplastic, metal or a composite material.
 17. The device as claimed inclaim 1, wherein the second hollow body comprises polypropylene.
 18. Akit for purifying or isolating nucleic acids from a sample comprisingthe device as claimed in claim
 1. 19. The kit as claimed in claim 18further comprising a chaotropic reagent.